Therapeutic drugs are typically administered orally or by intramuscular, subcutaneous, intraperitoneal, or intravenous injections. Intravenous injection is the most direct means of administration and results in the fastest equilibration of the drug with the blood stream. Drugs injected intravascularly reach peak serum levels within a short time, however. Toxic effects can result from such high serum levels, especially if the drug is given as a bolus injection. To avoid such high concentrations, drugs can be administered slowly as a continuous drip. This however requires prolonged nursing care and, in some cases, hospitalization which itself entails high cost. To avoid this, efforts have been made to develop means of administering drugs within stable carriers which allow bolus intravenous injections but provide a gradual release of the drugs inside the vasculature.
The reticuloendothelial system (RES) directs drugs preferentially to the liver and spleen, and its uptake of a carrier thus interferes with the distribution of the drug to other parts of the body. If however the carriers are small enough so that the phagocytic cells such as macrophages do not preferentially ingest them, the carriers would escape the RES long enough to perform other tasks. If the carriers also contain antibodies or other ligands on their surfaces which specifically bind to antigenic sites or specific receptors, these antibodies or ligands will direct the drugs to specific cell types containing these sites or receptors. This would result in a higher concentration of the drug near the surfaces of the targeted cells without a higher risk of systemic side effects.
Entrapment of useful agents serves useful purposes in other medical applications as well. Tiny air bubbles, for example, are useful in ultrasonography, where they are used to provide strong contrast to blood vessels and organs traversed by the bubbles. If the bubbles are injected through a peripheral vein, however, they must travel through the right heart, the pulmonary vasculature and then the left heart before they can reach to the other internal organs. Since the bubbles are inherently unstable, they are not able to remain small enough for effective ultrasonographic contrast by the time the intended organs are reached. Entrapment of small air bubbles in small particulate carriers would allow the bubbles to serve their intended function even after long distances of travel within the intravascular compartment.
Similar advantages by using small particulate carriers for contrast material for CAT scans and nuclear magnetic resonance (NMR) scans. Abnormally high concentrations of contrast material at an injection site which might lead to false interpretation of the results could be avoided by administration of the contrast material as an agent retained in a particulate carrier to be released later at the site of the organ of interest.
Oxygen is another vital biological molecule that can be carried within a particulate carrier if the carrier contains hemoglobin. While hemoglobin molecules in large amounts are toxic to the human body, entrapment of hemoglobin within a particulate carrier will reduce its toxicity to vital organs while permitting it to deliver oxygen.
To summarize, stable porous and membraneless carriers which deliver biological agents to sites within the body offer many advantages. The two major approaches of particulate carriers in the prior art are liposomes and microspheres.
In liposomes, a shell is formed by a lipid layer or multiple lipid layers surrounding a central hydrophilic solution containing the medication. The lipid layers are inherently unstable and much research went into stabilizing them during the manufacturing process. In addition, the lipid layer(s) may serve as a barrier to diffusion of certain molecules. It is difficult for a hydrophilic substrate to diffuse through the hydrophobic layers into the interior of the liposomes, or conversely, for the drugs to be released without physical destruction of the lipid layer(s).
Microspheres, in contrast to liposomes, do not have a surface membrane or a special outer layer to maintain their intactness. Most microspheres are more or less homogenous in structure. To maintain the stability of the microspheres, manufacturing procedures in the prior art include a cross-linking process to stabilize the microspheric mass. The cross-linking agent however alters the chemical nature of the natural biological molecule, which may render the resultant product antigenic to the injected host. An anaphylactic reaction to such a newly created antigenicity is unpredictable and potentially dangerous.
Various drugs such as aspirin and non-steroidal antiinflammatory drugs can affect platelet enzymes, which are important components in the circulation of blood within the body. These drugs can thus render the platelets ineffective in hemostasis. Other conditions like cancer or its treatments, including bone marrow transplants, also produce periods of thrombocytopenia which further aggravates the patient's condition. Trauma and surgical operations can lead to open blood vessels which leak not only erythrocytes, but also platelets. These and other conditions can lead to a weak platelet function and consequently impaired circulation.
While this can be remedied by platelet transfusion, certain problems can arise. These include immunization, bacterial and viral transmission, unavailability of platelets in the resuscitation facility because of short shelf-life, and high cost. Attempts to synthesize particulates that can serve as platelet substitutes are disclosed by Agam, G., et al., "Erythrocytes with covalently bound fibrinogen as a cellulare replacement for the treatment of thrombocytopenia," Eur. J. Clin. Invest. 22(2):105-112 (1992), and Coller, B.S., et al., "Thromboerythrocytes. In vitro studies of a potential autologous, semi-artificial alternative to platelet transfusions," J. Clin. Invest. 89(2):546-555 (1992).
It was known that during activation of platelets, fibrinogen molecules become bound to the surface of platelets. The cell recognition sequence within the fibrinogen molecules contains the sequence "arginine-glycine-aspartic acid (RGD)". Agam, et al. and Coller, et al. demonstrated that erythrocytes with covalently bound fibrinogen and various lengths of RGD, respectively, behaved as activated platelets in the presence of the host's natural platelets and can assist hemostasis.
These "thromboerythrocytes" are expected to have problems similar to those related to the transfusion of erythrocytes:
(a) the erythrocytes from which the final products are made have to be cross-matched to the recipient patient's blood type; PA1 (b) despite sensitive detection assays on the erythrocyte donor, there is no good way of assuring the absence of recent viral infection and viral inactivation on whole erythrocyte preparations are not practical; PA1 (c) the shelf-life of the product is limited by the storage time of erythrocytes; and PA1 (d) leakage of hemoglobin after injection of thromboerythrocytes may further aggravate the patient's condition from the problems associated with free hemoglobin infusions.
There is a need for platelet substitutes that do not have these problems.
Literature of potential relevance to the present invention is as follows.
U.S. Pat. No. 4,107,288, Oppenheim, et al., Aug. 15, 1978, for "Injectable Compositions, Nanoparticles Useful Therein, And Process of Manufacturing same" discloses a process of making microspheres of cross-linked macromolecules by using cross-linking agents such as an aldehyde hardening agent (glutaraldehyde cited as an example). In addition to the hardship in controlling the sizes of the microspheres formed, the Oppenheim process also produces many aggregations which are very undesirable for the purpose of an in vivo medication carrier.
U.S. Pat. No. 4,269,821, Kreuter, et al., May 26, 1981, for "Biological Material" discloses processes for the preparation of submicroscopic particles of a physiologically acceptable polymer associated with a biologically active material by using a cross-linking agent such as a polymerisable material soluble in a liquid medium (methyl methacrylate as an example).
U.S. Pat. No. 3,663,685, Evans et al., May 16, 1972, for "Biodegradable Radioactive Particles" (hereafter "Evans") discloses a method of preparing biodegradable radioactive particles by using heated water-oil solutions.
Widder, et al, "Magnetically Responsive Microspheres And Other Carriers For The Biophysical Targeting Of Antitumor Agents", Advances in Pharmacology and Chemotherapy 16:213-271 (1979) disclose emulsion polymerization methods of preparation of albumin microspheres (pages 233-235) and preparation of magnetically responsive albumin microsphere (pages 241-250). The methods essentially involve emulsification and heat denaturation of a water-oil solution to produce and stabilize microspheres. The authors also state that for heat sensitive drugs the microspheres are stabilized by chemical cross-linking.
To summarize this literature, typical prior art processes require irradiation, heat, or reaction with a cross-linking agent to polymerize the "monomers" (which are the individual protein molecules such as human serum albumin or gelatin molecules) to convert them to stable particles. Prior art methods which use heat to cross-link and stabilize the protein involve irreversible denaturation of the proteins which renders them "foreign" to the host body.
U.S. Pat. No. 5,049,322, Devissaguet, et al., Sep. 17, 1991 discloses a method of producing a colloidal system containing 150-450 nm particles by dissolving a protein ingredient in a solvent and adding ethanol or mixture of ethanol containing surfactant. Devissaguet does not disclose adding a second protein ingredient. Devissaguet discloses a process of producing colloidal spheres which have a distinct "wall" (column 2, line 25) or "layer" (column 8, line 33) of substance A which is different from the "core" of substance B (column 8, line 18), where the substance B may be a biologically active substance. This disclosure requires that the wall material and the core material both be present in a first liquid phase, which is then added to a second liquid phase that is a non-solvent for both materials. The resulting product is not homogeneous, and relies on the wall for its particle integrity.
Albert L. Lehninger, Biocheministry: The Molecular Basis of Cell Structure and Function (1972) discloses that ethanol as a solvent can decrease the ionization of proteins and therefore promote their coalescence and produce "colloidal suspensions". Lehninger does not disclose a special method of preparing colloidal suspensions, but rather generally a method of promoting protein coalescence by using ethanol, "s!ince a decrease in dielectric constant increases the attractive force between two opposite charges, ethanol decreases the ionization of proteins and thus promotes their coalescence" (page 134, lines 21 through 25, citations omitted). Lehninger has defined the process of "coalescence" as a process leading to "insoluble aggregates" (page 133, lines 31 through 35).
"Remington's Pharmaceutical Sciences", 7th ed. (1985) discloses some general knowledge of "colloidal dispersions". Remington teaches that adding surfactant "stabilizes the dispersion against coagulation" (page 286, column 2, lines 59 and 60), where the surfactant molecules "arrange themselves at the interface between water and an organic solid or liquid of low polarity in such a way that the hydrocarbon chain is in contact with the surface of the solid particle or sticks inside the oil droplet while the polar bead group is oriented towards the water phase" (page 286, column 2, lines 30 through 35). Remington does not specially disclose the use of any particular protein molecules such as globin as the primary protein.